Velos pro orbitrap elite

Mass spectrometry measurements were performed by using nano-ESI-FTMS. Samples were injected into nanospray capillaries from New Objective Inc. (Woburn, MA, USA) with an inner tip diameter of 2 mm and analyzed with a Velos Pro Orbitrap Elite mass spectrometer (Thermo Fisher Scientific Inc.,Waltham, MA, USA). The temperature of the transfer capillary was fixed at 240 °C. All measurements were performed in the positive ionization mode with an applied voltage to the nanospray capillary of 3 kV. The voltages applied to the S-Lens and multipoles were automatically tuned to favour the transmission of high m/z values. Full MS spectra were recorded using the Orbitrap analyzer, by averaging at least 110 scans in the range of m/z 2000–4000. Acquisition and treatment of the data was done with the Xcalibur software.

Approximately 3 g leaf tissues from 20‐d‐old 35S‐USL1‐MYC and 35S‐VPS30‐FLAG transgenic plants were ground in liquid N₂. The proteins were extracted and purified using the method described previously by Li et al. (2015). The purified proteins were separated using SDS‐PAGE. The entire gel lane was excised and dehydrated. Proteins were digested in‐gel with nedoproteinase trypsin (0.5 ng μl⁻¹ trypsin in 50 mM ammonium bicarbonate, pH 8.5). The extracted peptides were sequenced by LC‐MS/MS using a Velos Pro Orbitrap Elite mass spectrometer (Thermo Scientific, Waltham, MA, Waltham, MA, USA) equipped with a nano‐ESI source. The IPI (International Protein Index) Arabidopsis protein database was used as a searching platform.

High-resolution measurements were obtained with a VelosPro Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) fitted with a heated electrospray ionization source operating in positive and negative ionization modes. The mass spectrometer settings were as previously reported [116 (link)]. Collision-induced dissociation spectra (CID) in resonant excitation conditions were acquired using data-dependent scanning function for identification purpose. Non-targeted CID experiments using data-dependent scan were conducted at 27% Normalized Collision Energy (NCE) in positive and negative modes. CID experiments to confirm the presence of Pse (Leg) in metabolome were conducted at 22% NCE in positive and negative modes.

TAP purification of all constructs has been described previously [8 (link), 9 (link)]. Eluates were directly analyzed on a Velos-Pro Orbitrap Elite hybrid mass spectrometer (Thermo Fisher, Waltham, MA, USA). Raw MS data were searched with SEQUEST (Thermo Fisher) or COMET [41 (link)], and protein false discovery rate was set at 2.5%.

Peptide samples were loaded onto a first-dimension trap column (Waters Xbridge, C18, 10 uM particle size, 100 Å pore size, 4 cm packing length 150 uM column inner diameter). Online peptide separation coupled to MS/MS was performed with a 2D-nanoLC system (nanoAcquity UPLC system, Waters) and a Velos-Pro/Orbitrap-Elite hybrid mass spectrometer (ThermoFisher Scientific). Six discrete elutions were performed at 1.5 uL/min with 5mM ammonium formate pH 10 using increasing concentrations of ACN (1%, 3%, 6%, 15%, 25% and 44%) and diluted with 6 uL/min 0.1% formic acid (FA) prior to loading onto a second dimension trap column (Dr. Maisch ReproSil-Pur, C18, 5 uM particle size, 120 Å pore size, 4 cm packing length 150 uM column inner diameter) connected to an analytical column (Orochem Reliasil, C18, 3 uM particle size, 90 Å pore size, 20–25 cm packing length 50 uM column inner diameter) with an incorporated electrospray emitter. Peptide separation was achieved using a gradient from 3 to 80% (V/V) of ACN in 0.1% FA over 115 minutes at a flow rate of 200 nL/min. The mass spectrometer was operated in data-dependent mode using a Top 10 method. Full MS scans (m/z 300–2000) were acquired in the Orbitrap analyzer (resolution = 120,000), followed by high energy collision induced dissociation (HCD) MS/MS (fm/z 100–2000, resolution = 15,000) at a normalized collision energy of 35%.

To compare the proteomes of the U251N shCTRL and shCAPN2 cells, 10⁶ cells were collected and washed with DPBS. Proteins were extracted and desalted with S-Trap™ mini columns according to the manufacturer’s instructions (ProtiFi™). Proteins were digested with trypsin (V5111, Promega). Peptide concentrations were determined using Pierce™ BCA Protein Assay Kit. 25 µg of each sample were labelled with TMTpro™ 16plex Label Reagent Set (A44520, Thermo Scientific™) and pooled. 100 µg of the pooled sample were fractionated on a high pH liquid chromatography (HPLC, Agilent 1100 Series). 54 fractions were collected and concatenated to 18 samples which were submitted for LC–MS/MS analysis on a Velos Pro Orbitrap Elite™ (Thermo Scientific™) mass spectrometer coupled to an Easy nanoLC 1000 (Thermo Scientific™) with a 200 cm µPAC™ column (PharmaFluidics). Data were analyzed using MSFragger in Fragpipe 16.0 [51 (link)] and the UniProt human reference proteome database downloaded on the 14^th of June 2021 (20,845 protein entries including 245 potential contaminants). We allowed for two missed cleavages, a fragment and a precursor mass tolerance of 20 ppm each. A mass shift of 304.207146 Da was set for the N-term and lysine (K) representing the tandem mass tags (TMT), a mass shift of 57.0214560 Da for cysteine (C) representing carbamidomethylations and of 40.010600 Da for N-term acetylation.